[unreadable] The trigger of the autoimmune destruction of pancreatic beta cells in Type 1 diabetes is unclear. Several studies suggest that an increased intestinal permeability due to alteration of intestinal tight junctions (tj) can be involved in the pathogenesis of autoimmune diseases, including Type 1 diabetes. The intestinal epithelium is the largest mucosal surface providing an interface between the external environment and the mammalian host. Therefore, it is not surprising that when the integrity of the tj system is compromised an immune response to environmental antigens, including autoimmune diseases, may develop. For the past 10 years our studies have focused on the mechanism(s) of action of a protein elaborated by Vibrio cholerae, zonula occludens toxin (Zot) that affects the competency of intestinal tj (3,4). Our experiments using Zot as a tool to gain insights into the regulation of tj function led to the discovery of zonulin, a human eukaryotic Zot analogue, and to the definition of some of its physiological and pathological roles. We extended our findings to disease states, including Type 1 diabetes, characterized by a leaky gut and established the role of zonulin in their pathogenesis. We have applied genetic, biochemical, and biological techniques to establish the structural and functional requirements to activate the zonulin system. Our overall hypothesis is that a subgroup of subjects with Type 1 diabetes experiences a zonulin-dependent abnormal increase in intestinal permeability that causes non-self antigens to cross the intestinal barrier, so triggering an aberrant autoimmune response targeting the pancreatic beta cells. We have assembled a strong interdisciplinary team in which expertise in intestinal tj pathophysiology, cellular and molecular biology, pediatric gastroenterology and endocrinology, diabetology, biostatistics, and human genetics are blend together to offer the best resources to successfully address this hypothesis. The long-term objective of this proposal is to investigate possible links between zonulin-dependent chronic increase in intestinal permeability and the onset of Type 1 diabetes in order to develop strategies for the prevention of the disease. We will use the combination of an animal model of Type 1 diabetes and human studies in order to gain insights into Type 1 diabetes-associated tj dysregulation at the cellular and molecular levels (R21 component of the application). This phase will be followed by human studies performed in our GCRC aimed at developing strategies for the prevention of Type 1 diabetes (R33 component of the application), provided that our milestones will be achieved (Milestones component of the application). [unreadable] [unreadable]